Supercooling promoting agent

ABSTRACT

During the investigation of the mechanism that cellular water in woody plants growing in cold districts keeps liquid state at low temperature, the inventors have studied to identify the causative substances. As the results, the present inventors identified supercooling promoting agents in woody plants. 
     The supercooling ability of identified flavonoid glycoside and synthesized flavonoid glycoside with similar structure was tested. It was found that the supercooling promoting agent comprising these flavonoid glycosides enables to stably supercool bulk water at low temperature for long-term. The aqueous solution containing the supercooling promoting agent of the present invention is useful to store biological materials at low temperature.

FIELD OF THE INVENTION

The present invention relates to a supercooling promoting agentcomprising flavonoid glycosides and an antifreezing and vitrificationliquid containing the flavonoid glycosides.

BACKGROUND OF THE INVENTION

It is known that cellular water in growing woody plants in colddistricts keep liquid state at low temperature. It is considered thatwater in xylem parenchyma cell is supercooled down to −40° C. due to thephysical property of water, since water in xylem parenchyma cells isisolated from external environment as water droplets (Non-patentreference 1). In other words, cell walls surrounding xylem parenchymacells act as barriers preventing dehydration from the cells andpreventing penetration of extracellular ices into the cells whenextracellular ices are generated, and thus intracellular water behavesas water droplets isolated from external environment and is supercooled.

Furthermore, it is suggested that phenol compounds contained inover-wintering plants function as antifreezing substances (Non-patentreference 2).

Still furthermore, it has been disclosed that flavonoids are used forfreezing medium for culturing reproductive cells and the like (Patentreference 1) and for cooling liquid of internal combustion engine andthe like as ingredients of antifreezing liquid (Patent reference 2).

Additionally, as for flavonoid glycosides as a supercooling promotingagent of the present invention, a various type thereof are present inplants including trees and in substances derived from living bodies assecondary metabolites (Non-patent reference 3).

Patent reference 1: Japanese Patent Application Public Disclosure No.2000-500327 (WO97/14785)

Patent reference 2: International Publication WO2004/074397

Non-patent reference 1: Kagaku to Seibutsu, vol. 43, No. 5, 280-282(2005)

Non-patent reference 2: Kagaku to Seibutsu, vol. 37, No. 12, 778-780(1999)

Non-patent reference 3: “FLAVONOIDS Chemistry, Biochemistry andApplications” published in 2006 by CRC Press Taylor and Francis Group.

Problems to be Solved by the Invention

Although it is known that phenol compounds (flavonoids) may have asupercooling activity (Non-patent reference 2), it is not known thatflavonoid glycosides have a supercooling promoting activity (Non-patentreference 3).

The present inventors have studied the mechanism by which cellular waterin woody plants growing in cold districts keeps liquid state at lowtemperature and have studied to identify the causative agents. As theresults, the present inventors succeeded in identifying the supercoolingpromoting agent in woody plants.

Means to Solve the Problems

The present inventors focused attention on xylem parenchyma cells, whichkeep stably supercooled state down to −40° C. for long-term such asseveral weeks in nature, and tried to isolate active ingredients fromxylem of woody plants based on supercooling activity as an indicator. Asthe result, the inventors clarified that the causative substance toinduce stable supercooling state in the cells was flavonoid glycosides.Furthermore, the inventors found that flavonoids glycosides with similarstructure were significantly promotable supercooling ability of aqueoussolution based on the elucidated structural property and accomplishedthe present invention.

In other words, the present invention is a supercooling promoting agentcomprising a flavonoid glycoside represented by the following generalformula:

wherein

at least one of X¹ to X⁴ is a sugar residue in which a hydrogen isremoved from the reducing end of hemiacetal hydroxyl group ofmonosaccharides or oligosaccharides and others are hydroxyl group orhydrogen atom; and

R¹ to R⁶, which may be identical to or different from the other groups,are hydrogen atom, hydroxyl or methoxy group.

Moreover, the present invention is an antifreezing liquid prepared bydissolving the supercooling promoting agent in water or in an aqueoussolution containing additives as usage, wherein the antifreezing liquidcomprises equal to or more than 0.01 g/L of the supercooling promotingagent.

Still moreover, the present invention is a vitrification liquidcomprising equal to or more than 0.01 g/L of the supercooling promotingagent in a vitrification solution, wherein the vitrification solutioncomprises 20-100% by volume of a cryoprotective agent alone or acombination thereof and the remainder is water or aqueous solutioncontaining additives as usage.

Advantages of the Invention

The supercooling promoting agent of the present invention enables tosupercool water by the addition of a flavonoid glycoside derived fromliving bodies or synthesized; and can be applied for low temperaturestorage, for controlling frozen state and the like.

The supercooling promoting agent of the present invention enables tolower freezing point of water and substances containing water by about15° C. from natural freezing point of water. The supercooling promotingagent enables to stably supercool bulk water at low temperature forlong-term.

Furthermore, the supercooling promoting agent of the present inventiongenerates nonfreezing liquid by mixing with water, wherein thenonfreezing liquid is usable at about −15° C. and usable for storingbiological materials and the like at low temperature for long-term.

The supercooling promoting agent of the present invention enables to useas a freeze control agent, which regulates the size of frazil crystalsby freezing water; aqueous solution of additives as usage; or additivedissolved in substance including water. Addition of the substance lowersfreeze starting temperature due to supercooling and thus reduces thesize of frazil crystals formed. Therefore, the solution containing theadditive could be used as a freezing control agent, which changes theice size variously, in case of freezing by changing the cooling rate,composition or concentration of additives.

Moreover, it is possible to lower the concentration of vitrificationliquid by the addition of the supercooling promoting agent of thepresent invention to a vitrification liquid containing highconcentration of cryoprotective agent. Lowering the concentration ofvitirification liquid may reduce the toxicity caused by dipping invitrification liquid, produce glass bodies efficiently at ultra-lowtemperature such as liquid nitrogen temperature, and enables to storebiological materials in glass bodies at ultra-low temperature, whereinthe biological materials have been difficult of vitric storage.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the silica gel column chromatography of ethylacetate-soluble fraction of isolates from Cercidiphyllum japonicum tree(Katsura tree).

FIG. 2 shows the supercooling activity of silica gel columnchromatography fractions. The horizontal axis shows the temperature ofcupper plate loaded with liquid droplets and the vertical axis shows thepercentage of frozen liquid droplets.

FIG. 3 shows the high performance liquid chromatography loaded with theabove fractions number 9 and 10.

FIG. 4 shows the ¹H-NMR spectrum of an acetylated derivative of compound1.

FIG. 5 shows the ¹H-NMR spectrum of an acetylated derivative of compound2.

FIG. 6 shows the ¹H-NMR spectrum of an acetylated derivative of compound3.

FIG. 7 shows the ¹H-NMR spectrum of an acetylated derivative of compound4.

FIG. 8 shows the viability of cells, after pig liver fragments werestored in supercooling state, by dipped in the low temperature storageliquid (at −5° C. and at −8° C.) containing kaempferol-7-O-glucoside(K7G). The horizontal axis shows storage time (days) and the verticalaxis shows viability (%).

FIG. 9 shows the viability of cranberry shoot apexes (buds) stored in75% vitrification liquid at room temperature for a specified loadingtime (control batch), and that of the above apexes dipped in liquidnitrogen after that and thawed to room temperature (frozen batch). FIG.9A used vitrification liquid containing kaempferol-7-O-glucoside andFIG. 9B used that without kaempferol-7-O-glucoside. The horizontal axisshows the loading time (min) and the vertical axis shows viability (%).

DETAILED DESCRIPTION OF THE INVENTION

The supercooling promoting agent of the present invention, i.e. aflavonoid glycoside, is represented by the following formula:

wherein

at least one of X¹ to X^(4,) preferably X¹ and X², or X² and X⁴, morepreferably X¹ and X², most preferably X², is a sugar residue in which ahydrogen is removed from the reducing end of hemiacetal hydroxyl groupof monosaccharides or oligosaccharides. Naturally, only those residuescontaining sugar residues bound to X¹ and X², or X² and X⁴ by glycosidebonding are known. Together with the residues, those with glycosylatedseveral synthesizable hydroxyl groups (such as X³) are not excluded.

Additionally, a hemiacetal hydroxyl group is represented for example bya hydroxyl group bound to C1 carbon of the basic skeleton of thefollowing monosaccharide and disaccharide:

The monosaccharide includes glucose, mannose and galactose; andoligosaccharide includes rutinose, raffinose and the like; and a sugarresidue is preferably monosaccharide such as glucose, mannose andgalactose.

X¹ to X⁴ other than sugar residues are hydroxyl group or hydrogen atom;and preferably at least one of them is hydrogen atom, and morepreferably X³ is hydroxyl group and X⁴ is hydrogen atom.

R¹ to R⁶, which may be identical to or different from the other groups,are hydrogen atom, hydroxyl or methoxy group.

Among them, R¹ is preferably hydrogen atom or hydroxyl group, morepreferably hydrogen atom.

R⁴ is preferably hydrogen atom or methoxyl group, more preferablyhydrogen atom. R², R³, R⁶ and R⁶ are preferably hydrogen atom.

Since flavonoid glycosides of the present invention are contained in allkinds of living bodies such as woody plants, they might be isolated fromthe woody plants or substances derived from living bodies or might besynthesized.

As for the woody plants, it is considered that woody plants of colddistricts contain the supercooling promoting agents in abundance andsuitable to the present purpose. Such acicular tree includes, forexample, Larix kaempferi (Japanese larch), Thuja occidentalis (whiteceder), Taxus cuspidata (Japanese yew), Cryptomeria japonica (Japanesecedar), Abies homolepis (Nikko fir), Abies sachalinensis (Todo fir),Picea jezoensis (Yezo Spruce), Picea glehnii (Sakhalin Spruce), Pinusparviflora (Japanese white pine), Pinus strobus (white pine), Pinussylvestris (Japanese red pine), Pinus thunbergii (Japanese black pine)and the like. Also, broad leaf tree includes Betula platyphylla var.japonica (white birch), Populus sieboldii (Japanese aspen), Castaneacrenata (Japanese Chestnut), Sorbus commixta (Japanese Rowan), Styraxobassia (Fragrant snowbell), Quercus crispula (Japanese oak), Ulmusdavidiana var. japonica (Japanese elm), Cercidiphyllum japonicum(Katsura tree) and the like.

Said supercooling promoting agents are contained also in woody plantsgrowing in regions other than cold districts regardless of the amount ofthe contents. Said flavonoid glycosides can be isolated from not onlyxylem including sap wood and heart wood of the tree species but alsobarks, winter buds and leaves. Moreover, although it is considered thatthe supercooling promoting agents are present in living cells(parenchyma cells), it is possible that the agents are present atextracellular portion.

Additionally, the agents are stable and can be isolated from not onlyraw standing crop but also dead standing crop and timber stored forlong-term.

The supercooling promoting agent of the present invention showssupercooling activity of temperature range between −0.1° C. and −15.0°C.

The supercooling activity (or represented as inhibiting activity of icenucleus formation) is the value measured by the following method. Abuffer solution containing dead bacterial cells of ice nucleation activebacteria (Erwinia ananas) mixed with 0.5 mg/mL of a test sample isprepared. A large number of 2 μL droplets of the solution are put on acupper plate, which is temperature-controllable. Then, the cupper plateis cooled at the rate of 0.2° C./min, the number of frozen droplets arecounted macroscopically and the temperature, wherein 50% of droplets arefrozen, is defined as freezing point. The supercooling activity isdefined as the difference (° C.) between the freezing point of solutioncontaining test sample and ice nucleation active bacteria, and that ofsolution containing only ice nucleation bacteria and buffer solution.

The supercooling promoting agent (or supercooling substance) is definedas the substance, which has supercooling activity far exceeding theconcentration-dependent freezing point depression induced by adding lowconcentration (generally equal to or less than 1% by volume or byweight) of the substance to water.

Although general substances such as salts, sugars and sugar alcoholsshow increased supercooling activity of about two-fold of freezing pointdepression, supercooling promoting agents show supercooling activity ofmore than 10 times or some times more than 100 times.

The supercooling activity of the present flavonoid glycoside is superiorto the activity of the following other so called supercoolingsubstances.

1) The unidentified crude extract extracted from seeds of various plants(peach and others) shows supercooling activity of water in thetemperature range between −2.6° C. and −8.1° C. (Caple et al., (1983)Cryoletters, 4, 59-64). However, the value was obtained under thecondition that only silver iodide having low ice nucleation formingability was used and the cooling rate was 1° C./min, which is far rapidthan the cooling rate of the supercooling promoting agent of the presentinvention. Hence, the condition is apt to induce transient supercooling.

2) Eugenol extracted from clove and its allied substances showsupercooling activity of water in the temperature range between −0.2° C.and −2.5° C. (Kawahara and Obata (1996) J. Antibact. Antifung. Agents,24, 95-100). The concentration of additives is 1 mg/mL and the coolingrate was 1° C./min, which is far rapid than the cooling rate of thesupercooling promoting agent of the present invention. Hence, thecondition is apt to induce transient supercooling.

3) Hinokitiol and its allied substances show supercooling activity ofwater in the temperature range of between −0.4° C. and −2.1° C.(Kawahara et al., (2000) Biosci. Biotechnol. Biochem., 64, 2651-2656).

The concentration of additives is 10 mM and the cooling rate was 1°C./min, which is far rapid than the cooling rate of the supercoolingpromoting agent of the present invention. Hence, the condition is apt toinduce transient supercooling.

4) Chitin polysaccharide with 130 kDa isolated from bacteria showsupercooling activity of water in temperature range between −0° C. and−4.2° C. (Yamashita et al., (2002) Biosci. Biotechnol. Biochem., 66,948-954). The concentration of additives is 50 μg/mL and the coolingrate was 1° C./min, which is far rapid than the cooling rate of thesupercooling promoting agent of the present invention. Hence, thecondition is apt to induce transient supercooling.

5) Various antifreeze proteins show supercooling activity of water atthe maximum −7.8° C. (Duman (2002) J. Comp. Physiol., 172, 163-168).

However, the concentration of added nonfreezing protein at the maximumsupercooling promoting activity is not known and high concentration ofcitric acid (0.5 M) is added together with the proteins at the maximumactivity. Only the antifreeze protein promotes supercooling of −1.2° C.

The supercooling promoting agent of the present invention comprises theflavonoid glycoside.

Said flavonoid glycoside is typically used for aqueous solution and canbe used for antifreezing liquid, wherein the flavonoid is dissolved inwater typically at equal to or more than 0.01 g/L, preferably between0.01 and 30 g/L, more preferably between 0.01 and 10 g/L, still morepreferably between 0.1 and 1.0 g/L.

Said antifreezing liquid is obtainable by dissolving flavonoid glycosidetypically in water, which could be replaced with aqueous solutioncontaining additives as usage. Said additives include, for example,ingredients of culture medium of animal and plant cells and ingredientsof storage solution of biological materials. The concentration ofadditives in aqueous solution may be suitably determined as usage.

Furthermore, the antifreezing liquid may contain other supercoolingpromoting agents and cryoprotective agents. In the case ofcryoprotective agents, a cryoprotective agent alone or a combinationthereof may be included at the concentration range between 1 and 40% byvolume, preferably between 1 and 20% by volume.

A cryoprotective agent is defined as a substance reducing freeze-induceddamage by adding the substance to biological materials or aqueoussolution dipped therewith. All of cryoprotective agents show an effector a combination thereof selected from the effects includingconcentration-dependent freezing point depression, reduction of frazilice formation, reduction of increase in salt concentration in freezingmaterials and promotion of vitrification

Such cryoprotective agents include, for example, methanol, ethanol,acetamide, DMSO, formaldehyde, ethylene glycol, propylene glycol,glycerol, proline, glucose, sorbitol, sucrose, trehalose, polyethyleneglycol, dextran 10-150, PVP, albmin, ficoll, HES and the like.

Such antifreezing liquid can keep liquid state down to about −15° C. forlong-term (1 to 2 weeks) under the condition, wherein no cryoprotectiveagent is added, or additives such as cryoprotective agent are mixed at aconcentration (less than about 1% by weight) with almost no effect tofreezing point depression.

Although biological materials (plant or animal cells or tissues, eatableor ornamental fish and seafood, plant itself such as vegetable, or apart thereof) are usually stored in the antifreezing liquid less thanabout 5° C., long-term low temperature storage is possible withoutfreezing by dipping the biological materials in the antifreezing liquidat less than 0° C., particularly between about 0° C. and −15° C.

Said antifreezing liquid enables to reduce the size of ice crystal bylowering the freeze starting temperature based on supercooling, and isusable as a freeze control agent of medications and foods prepared byfreeze-drying by the use of only antifreezing liquid or by simultaneoususe of cryoprotective agents. Isolates (crude extract and others)isolated from biological materials such as trees as well as the abovesubstances are applicable similarly.

In contrast, liquid containing high concentration of the abovecryoprotective agent is called as “vitrification liquid”; and water doesnot generate crystals even at ultra-low temperature (i.e., liquidnitrogen temperature) and becomes a glass body (amorphous ice)(Ed. byTakao Niino “Plant ultralow storage manual” issued from NationalInstitute of Agrobiological Sciences, 2006).

Vitrification liquid is defined as solution, wherein the cryoprotectiveagents are contained by themselves or in combination thereof at theconcentration between 20 and 100% by volume, preferably between 40 and100% by volume, and the remainder is water. Solvent such as animal andplant cell culture medium could be used instead of the water.

For culture and storage of animal and plant cells, it is preferable tomix equal to or more than 30% by volume, particularly equal to or morethan 40% by volume of water and culture medium of animal and plantcells. PVS2, the vitrification liquid most frequently used, comprisesculture medium supplemented with 30% by volume of glycerol, 15% byvolume of ethylene glycol, 15% by volume of DMSO and 0.4 M sucrose. Thetype and concentration of culture medium is suitably changed dependingon culture and material to be stored.

In the present invention, the supercooling promoting agent (theabove-described flavonoid glycoside) of the present invention is addedtypically equal to or more than 0.01 g/L, preferably between 0.01 and 30g/L, more preferably between 0.01 and 10 g/L, further preferably between0.1 and 1.0 g/L in the vitrification liquid.

The above-described glass body may maintain amorphous state at less thanfreezing point of vitrification liquid, for example at equal to or lessthan −15° C., particularly between at −60° C. and −273° C., for exampleat liquid nitrogen temperature (77K).

In the case of freeze preservation by vitrification, generally thematerial for storage is subjected to pre loading treatment for shortterm at room temperature or at temperature equal to or more than 0° C.The pretreatment leads to dehydration of moisture content in thematerials by high concentration of vitrification liquid and concurrentlyreplace moisture content in materials with vitrification liquid. Hence,moisture content in the materials is vitrified without forming icecrystals on sinking the materials in liquid nitrogen. When biologicalmaterials such as plants are sunk in vitrification liquid and areplunged into liquid nitrogen, moisture inside and outside of biologicalmaterials is changed to glass bodies (amorphous ices). Sincevitrification state dose not induce damage by freezing, biologicalmaterials can be lead to freeze preservation in vitrification liquid atultra-low temperature.

The present invention is illustrated in the following Examples, butthese Examples are not intended to limit the scope of the presentinvention

EXAMPLE 1

Twigs are collected from Cercidiphyllum japonicum tree (Katsura tree)growing naturally at Sapporo area in Hokkaido. After the xylem tissuesof Katsura tree are fractionated by a pencil sharpener, frozen in liquidnitrogen and ground to pieces as small as possible by the use of amortar and a pestle. The obtained 3.7 Kg of ground products were soakedin 20 L of methanol for 2 weeks. The obtained crude extract wascentrifuged at 14,000G (Hitachi: HIMC CF15R), and the supernatant wasrecovered.

The supernatant was dried and the dried material (93.8 g) was dissolvedin 300 mL water. The aqueous suspension of the crude extract wascentrifuged at 14,000 G at 20° C., and the supernatant was recovered.After the supernatant (300 mL) and 600 mL ethyl acetate were mixed,water-soluble fraction and ethyl acetate-soluble fraction were separatedby the use of a separatory funnel and dried.

The supercooling activities of these samples were measured by thefollowing methods. After a buffer solution (50 mM potassium phosphatebuffer, pH 7.0) containing dead bacterial cells (Wako pure chemicalindustries Ltd.,) of ice nucleation active bacteria (Erwinia ananas)mixed with 0.5 mg/mL of a test sample, 2 μL droplets of the solution areput on a cupper plate, which is temperature-controllable. Then, thecupper plate is cooled at the rate of 0.2° C./min, the number of frozendroplets are counted macroscopically and the temperature, wherein 50% ofdroplets are frozen, is defined as freezing point. The difference (° C.)between the freezing point and that of the buffer solution was measured.Obtained supercooling activity was about −2° C. and about −4° C.,respectively, for the water-soluble fraction and ethyl acetate-solublefraction.

The dried ethyl acetate fraction, which showed the highest supercoolingactivity, was fractionated to about 30 fractions through a self-producedsilica gel column chromatography by the use of “hexan-2-propanol-water”and “chloroform-methanol-water” as the eluent. The silica gel columnchromatograph is shown in FIG. 1. Then, supercooling activity of thesubstance from each fraction was measured according to the similarmethod to the above-described one. As shown in FIG. 2, the fractions 9and 10 show the maximum supercooling activity.

The above obtained fractions 9 and 10 were analyzed by high performanceliquid chromatography (column: Wakosil 5C18HG, solvent:methanol:water=1:1, flow speed: 1 mL/min). As shown the result in FIG.3, peaks (1 to 7), which show the presence of 7 substances, wereobtained. Among these peaks, only peaks 4, 5, 6 and 7 showedsupercooling activity (hereinafter, these substances are called ascompound 1 to 4 (Cj4 to 7)), and the activities are −1.8° C. (compound1), −7.7° C. (compound 2), −0.2° C. (compound 3) and −2.5° C. (compound4), respectively.

For these 4 types of compounds, negative-HRFAB-MS analysis was performedby mass spectrometer (JMS-AX500: JEOL). The masses of these compoundswere 463.0893 (Cj4), 447.0942 (Cj5), 477.1038 (Cj6) and 447.0958 (Cj7),respectively and these molecular formulas were expected as C₂₁H₂₀O₁₂(Cj4), C₂H₂₀O₁₁ (Cj5), C22H₂₂O₁₂ (Cj6) and C₂₁H₂₀O₁₁ (Cj7),respectively.

Furthermore, these compounds were acetylated and the reaction productswere analyzed by high resolution nuclear magnetic resonance apparatus(BRUKER: AMX-500) on various types of one dimensional and twodimensional NMR spectra analysis. Acetylation reaction was performedaccording to the following procedures: about 10 mg of dried sample wasdissolved in 200 μL of methanol, then was added with 2 mL of aceticanhydride and 1 mL of pyridine, and the mixture was kept at 70° C. for1.5 hr. After the obtained acetylated derivatives were purified bypreparative TLC, they were dissolved in chloroform-d, and were subjectedto NMR spectrum analysis on ¹H-NMR, ¹³C-COM, DEPT, ¹H-¹H COSY, HMBC,HSQC.

Since all of the compounds showed characteristic UV spectra withabsorption peaks at 250 to 270 nm and 300 to 380 nm, the compounds wereexpected to have flavonol backbone. ¹H-NMR spectrum of each acetylatedderivative is shown in FIGS. 4 to 7.

When ¹H-NMR spectrum of compound 1 (Cj4) is compared to that of compound4 (Cj7), there are 8 signals (δ 1.92 to 2.45) attributed to acetyl groupand signals (δ 7.33, 7.93, 7.96) attributed to hydrogen bound to 2′, 5′and 6′ positions of B ring in compound 1. Based on the result and HMBCcorrelation, compound 1 was identified as quercetin-3-O-β-glucoside(FIG. 4). ¹H-NMR spectrum of an acetylated derivative of compound 2(Cj5) shows 7 signals (δ 1.92 to 2.45) attributed to acetyl groupsimilar to those of compound 4 (Cj7) and signals (δ 7.27, 7.84)attributed to hydrogen bound to 2′, 3′, 5′ and 6′ positions of B ringand 2 signals (δ 6.73, 7.01) attributed to hydrogen bound to aromaticring. Additionally, ¹H-NMR spectrum of an acetylated derivative ofconstituent sugar obtained from acetylated acid hydrolyzate of compound2 was identical to that of acetylated glucose (FIG. 5). Since there isHMBC correlation between hydrogen at 1 position of constituent sugar andcarbon at 7 position of aglycon, compound 2 was identified askaempherol-7-O-β-glucoside.

When ¹H-NMR spectrum of an acetylated derivative of compound 3 (Cj6) wascompared to that of compound 4 (Cj7), there are single hydrogen (δ 6.79)bound to aromatic ring and a signal (δ 4.01) of methoxyl group incompound 3. Based on the result and HMBC correlation, compound 3 wasidentified as 8-methoxykaempferol-3-O-β-glucoside (FIG. 6).

¹H-NMR spectrum of an acetylated derivative of compound 4 (Cj7) shows 7signals (δ 1.92-2.45) attributed to acetyl group, signal (δ 7.23, 8.04)attributed to hydrogen bound to 2′, 3′, 5′ and 6′ positions of B ringand 2 signals (δ 6.84, 7.30) attributed to hydrogen bound to aromaticring.

Moreover, presence of β-glucose residues (δ 3.60, 3.96, 5.04, 5.17,5.28, 5.53) was confirmed. HMBC correlation was observed betweenhydrogen bound to anomeric carbon of glucose and carbon at 3 position ofaglycon. Based on the above results, compound 4 was identified askaempferol-3-O-β-glucoside (FIG. 7).

Based on these results on mass spectrometric and NMR spectrometricanalysis, it was concluded that all these compounds are flavonoidglycoside, which is a glycoside with a glucose bound to aglycon, whereinthe aglycon is any one of quercetin, kaempferol and 8-methoxykaempferol.

Namely, the isolated supercooling promoting agent is flavonoid glycosiderepresented by the following formula:

wherein the number in the formula represents the number of the compound.

SYNTHETIC EXAMPLE 1

In the present synthetic example, compound 5(Chrycin-7-O-β-D-glucopyranoside) was synthesized. Chrycin (TokyoChemical Industry, Ltd., Tokyo, Japan, 0.51 g (2.0 mmol)), 1.38 g (10mmol) of K₂CO₃, 0.12 g (0.4 mmol) of benzyltributyl-ammoniumchloride andCHCl₃ (10 mmol) were stirred with a magnet, added with 1.61 g (3.8 mmol)of tetra-O-acetyl-α-D-glucopyranosilbromide (Kanto Chemical Co.), atroom temperature and were refluxed with heating for 2 hr. Furthermore,the mixture was added with above described bromide (1.00 g (2.4 mmol))and was kept refluxing with heating for 1 hr. After the reaction mixturewas shaken with 20 mL of 2N hydrochloric acid, organic layer wasseparated and dried with MgSO₄. The residue obtained by concentrationunder reduced atmosphere was crystallized in hexane-ethanol, and 1.03 gof Chrycin 7-O-β-D-tetra-O-acetylglucopyranoside was obtained (yield88%).

The analytical values of the product are shown as follows.

FAB-MS: m/z 585 (M+H⁺, 57%), 331 (29), 255 (100). FAB-HR-MS: m/z585.1599 (calc. for C₂₉H₂₈O₁₃+H⁺, 585.1609)

¹H NMR (DMSO-d6) δ1.97 (3H, s), 2.02 (9H, s), 4.11 (1H, br d, J=12.4),4.19 (2H, dd, J=5.3, 12.4), 4.35 (1H, m), 5.01 (1H, t like, J=9.6), 5.10(1H, dd, J=7.9, 9.6), 5.39 (1H, t like, J=9.6), 5.76 (1H, d, J=7.9),6.47 (1H, d, J=1.6), 6.84 (1H, d, =1.6), 7.08 (1H, s), 7.55-7.65 (3H,m), 8.09 (2H, d, J=7.9), 12.85 (1H, s, OH).

After the above obtained Chrycin 7-O-β-D-tetra-O-acetylglucopyranoside(0.23 g (0.39 mmol)) was added to 10 mL CH₃OH-Et₃N (2:1) and wasrefluxed with heating for 12 hr, it was concentrated and dried. Theobject (0.12 g (yield 72%)) was obtained by recrystallization of crudecrystal from hexane-ethanol. The analytical values of the product(compound 5) are shown as follows.

FAB-MS: m/z 417 (M+H⁺, 15%), 307 (32), 255 (34), 154 (100). FAB-HR-MS:m/z 417.1180 (calc. for C₂₁H₂₀O₉+H⁺, 417.1182)

¹H NMR (DMSO-d₆) δ3.1-3.6 (5H, m), 3.70 (1H, m), 4.62 (1H, br s, OH),5.08 (2H, d like, J=6.9, anomeric H, OH), 5.15 (1H, br s, OH), 5.43 (1H,br s, OH), 6.47 (1H, d, J=2.0), 6.87 (1H, d, J=2.0), 7.06 (1H, s),7.45-7.70 (3H, m), 8.09 (2H, d, J=6.5), 12.72 (1H, br s, OH).

¹³C NMR (DMSO-d₆) δ60.5, 69.5, 73.0, 76.4, 77.1, 94.9, 99.6, 99.7,105.4, 105.5, 126.4, 129.0, 130.4, 132.1, 156.9, 160.9, 163.0, 163.5,182.0.

SYNTHETIC EXAMPLES 2 AND 3

In the present synthetic example, compound 6 (apigenin 7-O-62-D-glucopyranoside) and compound 7 (apigenin 4′,7-di-O-β-D-glucopyranoside) were synthesized.

(1) Synthesis of apigenin 7-O-β-D-tetra-O-acetylglucopyranoside andapigenin 4′, 7-di-O-β-D-tetra-O-acetylglucopyranoside.

According to the previous report (J. Chin. Chem. Soc., 48, 201-206(2001)), apigenin was prepared by iodating naringenin (Tokyo ChemicalIndustry, Ltd.). Apigenin (1.66 g (6.1 mmol)), 3.59 g (9.2 mmol) oftetra-O-acetyl-α-D-glucopyranosilbromide (Kanto Chemical Co.) and 2.54 g(9.2 mmol) of Ag₂CO₃ were added to 30 mL of quinoline-pyridine (1:1) andstirred at room temperature for 1 hr. Furthermore, the above bromide(1.21 g (3.1 mmol)) and 0.83 g (3.0 mmol) of Ag₂CO₃ were added to theabove mixture and subjected to further reaction for 8 hr. The reactionmixture was diluted in 50 mL of acetone and filtrated through Celiteafter stirring. After the residue obtained by concentration of thefiltrate under reduced pressure was redissolved in 100 mL ofethylacetate, and shaken with 30 mL of 2N hydrochloric acid and thenwith saturated sodium chloride solution, the separated organic layer wasdried (MgSO₄). The remainder obtained by concentration under reducedpressure was subjected silica gel column chromatography(chloroform:methanol=40:1 and chloroform:ethyl acetate=1:1) for twotimes. As the result, apigenin 7-O-β-D-tetra-O-acetylglucopyranoside(1.00 g (yield 27%)) and apigenin 4′,7-di-O-β-D-tetra-O-acetylglucopyranoside (0.21 g (yield 4%)) wereobtained. The analytical value of the product is shown as follows.

(a) Apigenin 7-O-β-D-tetra-O-acetylglucopyranoside;

FAB-MS: m/z 601 (M+H⁺, 30%), 331 (41), 271 (91), 169 (100). FAB-HR-MS:m/z 601.1541 (calc. for C₂₉H₂₈O₁₄+H⁺, 601.1558)

¹H NMR (DMSO-d₆) δ1.97 (3H, s), 2.02 (9H, s), 4.11 (1, br d, J=12.5),4.19 (1H, dd, J=5.3, 12.5), 4.33 (1H, m), 5.01 (1H, t like, J=9.7), 5.09(2H, dd, J=6.9, 9.7), 5.39 (1H, 9.7), 5.74 (1H, d, J=7.9), 6.44 (1H, d,J=2.1), 6.79 (1H, d, J=2.1), 6.89 (1H, s), 6.92 (2H, d, J=8.7), 7.95(2H, d, J=8.7), 13.02 (1H, s, OH).

(b) Apigenin 4′, 7-di-O-β-D-tetra-O-acetylglucopyranoside;

FAB-MS: m/z 931 (M+H⁺, 44%), 601 (33), 271 (66), 169 (49), 43 (100).FAB-HR-MS: m/z 931.2524 (calc. for C₄₃H₄₆O₂₃+H⁺, 931.2508)

¹H NMR (DMSO-d₆) δ1.97 (6H, s), 2.01 (18H, s), 4.0-4.25 (4H, m),4.25-4.37 (2H, m), 4.95-5.15 (4H, m), 5.3-5.5 (2H, m), 5.76 (2H, br d,J=7.9, anomeric H), 6.47 (1H, d, J=2.0), 6.83 (1H, d, J=2.0), 7.05 (1H,s), 7.17 (2H, d, J=8.9), 8.10 (2H, d, J=8.9), 12.91 (1H, s, OH).

(2) Synthesis of compound 6 (apigenin 7-O-β-D-glucopyranoside) andcompound 7 (apigenin 4′, 7-di-O-β-D-glucopyranoside)

Apigenin 7-O-β-D-tetra-O-acetylglucopyranoside (0.21 g (0.35 mmol)) asobtained above was added to 10 mL CH3OH-Et3N (2:1), refluxed withheating for 12 hr, and then concentrated and dried. The object (0.11 g)was obtained by recrystallization of obtained crude crystals frommethanol (yield 73%).

Apigenin 4′, 7-di-O-β-D-glucopyranoside (78 mg) was obtained from 0.17 gof apigenin 4′, 7-di-O-β-D-tetra-O-acetylglucopyranoside as obtainedabove by the similar reaction (yield 71%). The analytical value of theproduct is shown as follows.

(c) Compound 6 (apigenin 7-O-β-D-glucopyranoside)

FAB-MS: m/z 433 (M+H⁺, 9%), 241 (96), 185(100). m/z 431 (M−H⁺, 7%), 279(20), 269 (24), 148 (100).

FAB-HR-MS: m/z 431.0993 (calc. for C₂₁H₂₀O₁₀—H⁺, 431.0978)

MS (FAB⁺): m/z 433 (M+H⁺), 185, 150, 93, 75, 57, 45.

¹H NMR (DMSO-d₆) δ3.0-3.6 (5H, m), 3.70 (1H, dd, J=4.8, 9.4), 4.60 (1H,m, OH), 5.08 (2H, d like, J=5.1, anomeric H, OH), 5.13 (1H, d, J=4.5,OH), 5.39 (1H, d, J=4.5, OH), 6.43 (1H, d, J=2.1), 6.82 (1H, d, J=2.1),6.89 (1H, s), 6.93 (2H, d, J=8.8), 7.95 (2H, d, J=8.8), 10.40 (1H, hr s,OH), 12.95 (1H, s, OH).

¹³C NMR (DMSO-d₆) δ60.6, 69.5, 73.0, 76.4, 77.1, 94.7, 99.4, 99.8,103.0, 105.2, 115.9, 120.9, 128.5, 156.7, 160.9, 161.2, 162.7, 164.0,181.8.

(d) Compound 7 (apigenin 4′, 7-di-O-β-D-glucopyranoside)

FAB-MS: m/z 595 (M+H⁺, 0.6%), 271 (4), 185 (56), 93 (100). FAB-HR-MS:m/z 595.1688 (calc. for C₂₇H₃₀O₁₅+H⁺, 595.1663)

¹H NMR (DMSO-d₆) δ3.1-3.6 (10H, m), 3.70 (2H, m), 4.55-4.65 (2H, m, OH),5.0-5.1 (4H, m, anomeric H×2, OH×2), 5.14 (2H, d like, J=4.0, OH),5.35-5.45 (2H, m, OM, 6.44 (1H, d, J=2.1), 6.86 (1H, d, J=2.1), 6.99(1H, s), 6.19 (2H, d, J=8.9), 8.06 (2H, d, J=8.9), 12.88 (1H, s, OH).

¹³C NMR (DMSO-d₆) δ60.55, 60.60, 69.5, 69.6, 73.0, 73.1, 76.4, 76.5,77.1, 94.8, 99.5, 99.7, 104.0, 105.3, 116.5, 123.5, 128.1, 156.8, 160.2,160.9, 162.8, 163.4, 181.8.

SYNTHETIC EXAMPLE 4

In this synthetic example, compound 8 (rhoifoline, apigenin7-O-β-neohesperidoside) was synthesized.

Naringin dihydrate (Tokyo Chemical Industry, Ltd., 1.23 g (2.0 mmol))and 0.51 g (2.0 mmol) of iodide were added to pyridine (5 mL), andrefluxed with heating for 9 hr under stirring. After the reactant wasleft to room temperature, insoluble matters were filtered off and thefiltrate was concentrated under reduced pressure. The object (0.47 g)was got by crystallization of the obtained remainder from watercontaining ethanol (yield 41%). The analytical value of the product isshown as follows.

FAB-MS: m/z 579 (M+H⁺, 10%), 277 (57), 241 (63), 207 (72), 185 (100).FAB-HR-MS: m/z 579.1712 (calc. for C₂₇H₃₀O₁₄+H⁺, 579.1714)

¹H NMR (DMSO-d₆) δ1.19 (3H, d, J=6.1), 3.1-3.9 (9H, m), 4.47 (1H, d,J=4.4, OH), 4.6-4.75 (3H, m, OH ×3), 5.12 (1H, s, anomeric H), 5.16 (1H,d, J=4.4, OH), 5.22 (H, d, J=6.6, anomeric H), 5.34 (1H, d, J=4.4, OH),6.36 (1H, br s), 6.78 (1H, br s), 6.86 (1H, s), 6.93 (2H, d, J=8.6),7.93 (2H, d, J=8.6), 10.40 (1H, s, OH), 12.96 (1H, s, OH).

¹³C NMR (DMSO-d₆) δ18.1, 60.4, 68.3, 69.6, 70.35, 70.43, 71.8, 76.2,77.0, 77.1, 94.4, 97.7, 99.2, 100.4, 103.1, 105.3, 115.9, 120.9, 128.4,156.8, 160.9, 161.2, 162.3, 164.0, 181.7.

EXAMPLE 2

The supercooling activities of flavonoid glycoside (the followingcompound 5 to 8, wherein the number in the formula represents the numberof the compounds) synthesized in synthetic examples 1 to 4 were measuredby the method similar to example 1, except that the concentration of thetest compounds was 0.1 mg/mL.

Resultantly, the supercooling activity of compound 5(Chricin-7-O-D-glucopyranoside), that of compound 6 (apigenin7-O-D-glucopyranoside), that of compound 7 (apigenin 4′,7-di-O-D-glucopiranoside) and that of compound 8 (rhoifolin) are −4.5°C., −12.0° C., −4.9° C. and −1.8° C., respectively.

EXAMPLE 3

The storage liquid was prepared by adding 0.01% by weight ofkaempferol-7-glucoside (compound 2 (Cj5)), Extrasynthese Co.) and 1% byvolume of DMSO (Wako pure chemical industries Ltd., guaranteed reagent)to a buffer solution (UW liquid, 100 mM lactobionic acid, 25 mM KH2PO4,5 mM MgSO₄, 30 mM raffinose, 2.5 mM adenosine, 3 mM GSH, 1 mMallopurinol, 0.25 mg/mL streptomycin, 10 UI/mL penicillin).

Although, DMSO was used to enhance the solubility ofkaempferol-7-O-glucoside, there was no effect on viability even for 4°C. storage sample (FIG. 8).

For comparison, a mixed solution (UW liquid) withoutkaempferol-7-O-glucoside and DMSO was prepared.

The viability of cells was evaluated by trypan blue (GIBCO) staining,when pig liver cells 5×106 cells/mL were dipped in 0.5 cc of the abovestorage liquid, supercooled at −5° C. and at −8° C., and stored for 1, 4and 7 days.

The result is shown in FIG. 8. It was found that low temperature storageby supercooling induced by kaempferol-7-O-glucoside enables storage ofviable animal cells for long-term.

EXAMPLE 4

As a vitrification liquid, 75% dilution solution of PVS2 liquid (22.5%by volume glycerol, 11.25% by volume of ethyleneglycol, 11.25% by volumeof DMSO, 0.4 M sucrose, the remainder was Murashige & Skoog medium,DUCHEFA BIOCHEMIE By) supplemented with 0.05% by weight ofkaempferol-7-O-glucoside (compound 2, Extrasynthese Co.) was used(hereinafter referred to as “75% vitrification liquid”). Since viabilityof cranberry shoot apexes is lowered significantly with loading time bydipping in PVS2 liquid at room temperature due to chemical toxicity, themixed solution with reduced content (75%) of vitrification liquid asdescribed above was used.

For comparison, a mixed solution without kaempferol-7-O-glucoside wasprepared. Shoot apexes (buds) are isolated from cranberry (subculturedin Hokkaido University, Graduate School of Agriculture) and dipped inthe above mixed solution at room temperature.

Since the concentration of vitrification liquid was reduced, viabilityof apexes as a function of loading time at room temperature wascomparatively high in spite of the gradual decrease (control batches ofFIG. 9A and FIG. 9B).

On the other hand, survival of shoot apexes, which were dipped in 75%vitrification liquid containing kaempferol-7-O-glucoside at roomtemperature for a specified loading time, put into liquid nitrogen,frozen for overnight, and then thawed at room temperature, is shown infrozen batch of FIG. 9A. Similarly, survival of the apexes, which weredipped in a vitrification liquid without kaempferol-7-O-glucoside andfrozen, is shown in frozen batch of FIG. 9B.

The survival of apexes frozen in vitrification liquid without mixingkaempfrol-7-O-glucoside shows significant damage due to freezing (frozenbatch of FIG. 9B).

Since addition of kaempferol-7-O-glucoside to vitrification liquidsignificantly reduced the damage due to freezing (frozen batch of FIG.9A), it verified that supercooling storage was possible.

The vitrification storage of the biological materials such as cranberryshoot apexes has been impossible due to chemical toxicity ofconventional vitrification liquid. However, addition of supercoolingpromoting agent of the present invention enabled to use as vitrificationliquid even if the concentration of vitrification liquid was lowereddown to the concentration, wherein the chemical toxicity is negligible,and enabled to store the biological materials in frozen state at extremecold state without damage by chemical toxicity due to high concentrationof vitrification liquid and by freezing.

1-8. (canceled)
 9. A medication containing as a freeze control agent aflavonoid glycoside represented by Chemical Formula 1:

wherein at least one of X¹ to X⁴ is a sugar residue in which a hydrogenis removed from the hemiacetal hydroxyl group of monosaccharides oroligosaccharides and others are hydroxyl group or hydrogen atom; and R¹to R⁶, which may be identical to or different from the other groups, arehydrogen atom, hydroxyl or methoxy group.
 10. The medication of claim 9,wherein the medication is prepared by freeze-drying.
 11. A foodcontaining as a freeze control agent a flavonoid glycoside representedby Chemical Formula 1:

wherein at least one of X¹ to X⁴ is a sugar residue in which a hydrogenis removed from the hemiacetal hydroxyl group of monosaccharides oroligosaccharides and others are hydroxyl group or hydrogen atom; and R¹to R⁶, which may be identical to or different from the other groups, arehydrogen atom, hydroxyl or methoxy group.
 12. The food of claim 11,wherein the food is prepared by freeze-drying.
 13. A method forcontrolling the freezing of a sample comprising: a) providing a samplecontaining a flavonoid glycoside represented by Chemical Formula 1:

wherein at least one of X¹ to X⁴ is a sugar residue in which a hydrogenis removed from the hemiacetal hydroxyl group of monosaccharides oroligosaccharides and others are hydroxyl group or hydrogen atom; and R¹to R⁶, which may be identical to or different from the other groups, arehydrogen atom, hydroxyl or methoxy group; and b) freeze-drying thesample.
 14. The method of claim 13, wherein the sample is a food. 15.The method of claim 13, wherein the sample is a medication.